Bacterial resistance to antibiotics is becoming a major threat to public health. It is imperative to find new therapeutic interventions to fight pathogens. Thus, deciphering host-pathogen interactions may allow defining targets for new strategies for effective treatments of infectious diseases. This chapter focuses on the bacterial manipulation of the host cell actin cytoskeleton. We discuss three infectious processes. The first is pathogen establishment of infection/invasion, explaining cellular uptake pathways that rely on actin, such as phagocytosis and macropinocytosis. The second process focus on the establishment of a replication niche, a process that subverts cytoskeletal functions associated with membrane trafficking namely phagosome maturation and cellular innate immune responses. Finally, pathogen dissemination is an emerging field that microfilaments have shown to participate: pathogen motility through the cytoplasm and from cell-to-cell or on the outer surface of the plasma membrane mimicking a receptor tyrosine kinase signaling pathway that helps the projection of pathogens to neighboring cells. It also establishes a connection with the innate immunity related with induction of cell signaling to inflammation, inflammasome activation, and programmed cell death. These studies revealed several potential targets related to actin cytoskeleton manipulation to design new therapeutic strategies for bacterial infections.
- Rho GTPases
- bacterial pathogens
- virulence mechanisms
- innate immunity
The cell cytoskeleton is composed of three distinct protein families each of which is assembled from monomers to form polymer networks namely from actin, tubulin, or intermediate-filament proteins. Host and pathogens have developed intrinsic interactions with the cytoskeletal system, playing a central role in several stages of their life cycles. Deciphering the complexity of these interactions is revealing new insights about the mechanisms of bacterial pathogenicity but also on defining new host targets for alternative therapies to available antibiotics. Indeed, clarifying these bacterial mechanisms of host subversion has led to many discoveries about host cell biology, including the identification of new cytoskeletal proteins, regulatory pathways, and mechanisms of cytoskeletal function. Microorganisms exploit actin, microtubules, and intermediate filaments in diverse ways, however, it is mainly the actin cytoskeleton that appears to play a critical role in infection and is the topic of this chapter.
In host cells, actin is involved in the polymerization of stable filaments to assure the cell architecture; at the cell surface originates dynamic movements mediated via assembly and disassembly of microfilaments contributing to contour changes as well cellular locomotion, cell-to-cell adhesion, and signaling. In the cytoplasm, the actin skeleton provides tracks and tails to direct vesicle trafficking. Thus, the importance of the actin cytoskeleton for eukaryotic host physiology from cell movement, cell-to-cell adherence, endocytosis, vesicle trafficking, and cell signaling, among others, has provided pathogenic bacteria with a plethora of opportunistic chances to be exploited.
The roles of the actin cytoskeleton in host-pathogen interactions can be summarized according to groups of pathogens and how they interact with this system. Some promote attachment to the plasma membrane, forming specialized actin structures (pedestals), allowing strong adherence to host epithelial surfaces. Others induce actin polymerization to enter into nonprofessional phagocytic cells; while others prevent polymerization to avoid uptake by professional phagocytic cells. A few pathogens use the actin cytoskeleton to allow other specialized internalization processes to occur in phagocytic cells as an alternative or in addition to phagocytosis. Intracellular pathogens manipulate the cytoskeleton to prevent membrane trafficking or fusion events leading to the establishment of a niche inside a vacuole often avoiding delivery into the degradative environment of the lysosome. Finally, some pathogens escape from the phagosome vacuole to the cytosol and use the actin machinery to move within cells and to spread directly from the cytoplasm of one cell into the cytoplasm of an adjacent cell. Recently, actin dynamics during infection was related to innate immune responses that rely on activation of cytosolic pattern recognition receptors (cytosolic PRRs) for inflammasome or autophagy assembly and programmed cell death.
This chapter provides a comprehensive summary of various strategies used by both extracellular and intracellular bacteria to hijack the host actin cytoskeleton (Figure 1).
2. Acting on actin during pathogen establishment of infection/invasion
Pathogens often have to overcome epithelial barriers to gain entry into the host cells. The first of which is the epithelial mucosae and a few pathogens, along their evolution, have developed strategies to overcome these barriers by means of active invasion mechanisms. Therefore some intracellular pathogens have evolved strategies to induce or modulate their uptake into these nonprofessional phagocytic cells. Alternatively, as a barrier circumventing mechanism, they may use the cells of the immune system (professional phagocytic cells such as macrophages, neutrophils, and dendritic cells) that patrols those epithelia. Here pathogens may or not play an active role in host cell internalization. Usually professional phagocytes recognize pattern signatures of pathogens (e.g., lipopolysaccharides: LPS), or opsonized bacteria (e.g., complement C3 or IgGs), by means of surface receptors. Likewise phagocytes play an active role in bacteria internalization. As part of the immune system these cells are equipped with a series of insult mechanisms designed to clear pathogens (as the proteolysis at low pH in the phagolysosome). Likewise, extracellular pathogens modulate the host cell plasma membrane for attachment and inhibition of phagocytosis in order to survive. In contrast, intracellular pathogens developed strategies to circumvent the bactericidal mechanisms of immune cells via establishing a protective vacuolar niche.
Several actin dependent mechanisms exist for allowing the establishment of infection: (1) Conventional phagocytosis meaning the entry into professional phagocytes by bilateral membrane pseudopodia formation that tightly encloses the bacteria. Phagocytosis always involves close contact between particle and plasma membrane by multivalence receptor-ligand interactions following morphological changes assembling a zipper mechanism. The host plays a central role for the internalization event while no action is required from the pathogen; (2) induced phagocytosis, a process of active induction of internalization into nonprofessional phagocytes such as epithelial cells, by pathogen manipulation of the host cell contractile system; both the host and the pathogen have active roles in the event. Mechanistically the process occurs by strong interactions between bacterial ligands with cell receptors as in conventional phagocytosis; (3) macropinocytosis: here there may be no direct contact between ligand-pathogen and cell-receptors. Literally, macropinocytosis means—cell drinking—and always involves extensive signaling (e.g., via EGF receptor, a type of tyrosine kinase receptor) that induces pseudopodia unilateral formation surrounding large amount of extracellular volume. So particles including bacteria go in passively along with extracellular fluid. Conventional macropinocytosis may occurs in several types of cells including professional and nonprofessional phagocytes leading to the formation of a large vacuole, the macropinosome; (4) induced macropinocytosis involves pathogen manipulation of the host cell cytoskeleton through growth factor induced signaling or directly using secretion systems that injects virulence factors into the cytosol. While referred classically as trigger phagocytosis, according to the type of morphological changes (with multiple ruffles at the cell surface), there is no direct connection between pathogen and plasma membrane. Finally, (5) an unconventional form of phagocytosis may be used for the establishment of infection via actin cytoskeleton. This is termed as coiling phagocytosis and involves single folds of the phagocyte plasma membrane wrapping around microbes in multiple turns (Figure 1).
2.1. Phagocytosis of bacteria and inhibition of phagocytosis by pathogens
Phagocytosis is a universal phenomenon involving the recognition and binding of a particle (over 0.5 μm in diameter), in a multivalence receptor-dependent manner, to its internalization and degradation within the phagocytic cell . Mechanistically the process of particle internalization from the plasma membrane is clathrin independent and requires actin polymerization . Phagocytosis of one particle does not signal or permit the indiscriminate phagocytosis of other particles bound to the cell surface. In fact particle ingestion is not automatically triggered by initial particle binding, but requires the sequential recruitment of cell surface receptors into interactions with the remainder of the particle surface. The forming phagosome conforms to the shape of the particle as a close-fitting sleeve of plasma membrane, held in place by interactions between surface receptors and the particle surface, much as teeth hold a zipper together . Phagocytosis can be broadly categorized into three steps: particle binding (along with receptor-cell signaling), internalization (i.e., phagosome formation and invagination) and phagosome maturation (i.e., biogenesis of the degradative compartment: the phagolysosome).
The phases prior to the establishment of interactions between bacterial ligands and phagocytic receptors may involve pathogen fishing by cell structures—this process is also dependent of filamentous actin (F-actin), filopodia extensions (Figure 1). Filopodia serves differently in pathogens and immune cells: pathogens will use it to approach cell membranes for invasion while macrophages will take advantage of these structures for fishing surrounding molecules in order to patrol the environment for possible invaders .
Phagocytosis was first discovered in the lower eukaryote amoebae that use it for feeding. In higher organisms, phagocytosis is fundamental for host defence against invading pathogens and contributes to the immune and inflammatory responses  including turnover and remodeling of tissues and disposal of dead cells. All cells may to some extent perform phagocytosis . However in mammals, phagocytosis is the hallmark of specialized cells including macrophages, dendritic cells, and polymorphonuclear neutrophils—these cells are collectively referred to as professional phagocytes . In certain circumstances, other cell types, such as fibroblasts engulfing apoptotic cells and bladder epithelial cells consuming erythrocytes, are able to perform conventional phagocytosis as efficiently as professional phagocytes .
Professional phagocytes express a series of cell surface receptors which recognize a variety of microbial ligands. Receptors on the surface of the phagocytic cell orchestrate a set of signaling events that are required for particle internalization. However, most pathogens possess many different ligands on their surface. Their phagocytic uptake occurs via multiligand interactions, which induce the engagement of many receptors at the same time.
Two major categories of receptors involved in pathogen recognition are opsonic receptors and nonopsonic receptors (pattern-recognition receptors: PRRs) . Receptors for opsonins such as IgG antibodies and the complement fragment C3bi engage Fc
Bacteria opsonized by complement C3b, by IgG or having lipoarabinomannans at the cell wall surface will be recognized by complement receptors such as CR1 and CR3/4, Fc receptors or Man-6P receptors respectively, each triggering phagocytosis without stimulating a strong superoxide burst. The entry via these phagocytic receptors leads to the maturation of the forming phagosome into a very degradative lysosomal compartment that will destroy microbes . All these receptors will be downregulated during phagocyte activation either through bacterial proinflammatory components as in the case of LPS or cytokines as IFNγ .
Activated macrophages will in turn reprogram their expression profile in order to increase the ability to kill pathogens via oxidative bursts and decrease protein digestion extension from amino-acids to small peptides, for antigen presentation .
Phagocytosis uses the actin cytoskeleton to construct a cup and close the cup by contractile activities . Latter along phagosome maturation the actin cytoskeleton is also utilized for vesicle trafficking and fusion along the endocytic pathway . The induced polymerization of filamentous actin (F-actin) from globular actin (G-actin) beneath the site of attachment of the particle is the driving force behind ingestion and proceeds from signal transduction downstream of the phagocytic receptors . The precise signaling cascades linking activated receptors to actin polymerization are not fully understood yet it is well known that Rho GTPase family plays critical roles in controlling these cytoskeletal rearrangements . These, RhoA, Rac1, and cell division cycle 42 (Cdc42) act as molecular switches in controlling actin dynamics by regulating the actin-related protein 2/3 (Arp2/3) complex . Arp2/3 requires activation by nucleation-promoting factors, such as the Wiskott-Aldrich syndrome protein (WASP) family. Nucleation-promoting factors exist in an autoinhibited conformation until activated by Cdc42 and Rac1, as well as by phosphoinositide (PI) signaling (discussed latter in this chapter). Effectors such as Cdc42 and the phosphoinositide 4,5-bisphosphate PI(4,5)P2 (PIP2) synergize to activate WASP homolog N-WASP which triggers actin polymerization via Arp2/3 . As the newly formed actin branch grows, the plasma membrane is forced out, extending the membrane as pseudopodia (Figure 1).
Various extracellular and intracellular cues including those from pathogens stimulate Rho GTPases, leading to actin-mediated membrane manipulation. RhoA, Rac1, and Cdc42 have all been shown to accumulate at the nascent phagosome cup. These proteins are preferred targets for bacterial toxins that in turn modulate the organization of the actin skeleton allowing invasion into nonprofessional phagocytic cells and preventing phagocytosis into professional phagocytes. These toxins modify the activity of Rho GTPases through covalent modification or regulation of the nucleotide state. Toxins such as
Another group of toxins regulates the nucleotide state and thus the function of various Rho GTPases by acting as GTPase-activating proteins (GAPs).
Pseudomonas has the capacity to inactivate all Rho GTPases .
Many intracellular bacterial pathogens have evolved to survive and even proliferate within immune phagocytic cells
The uptake of
The heterodimeric host surface receptor complement-receptor 3 (CR-3), mediates uptake of opsonized and nonopsonized mycobacteria. Interestingly, CR-3 is targeted by other intracellular pathogens, such as
Lipid modification by receptor signaling creates the potential for radiating signals that can affect large areas of the plasma membrane. Phospholipid kinases, lipid phosphatases, and hydrolases are activated during phagocytosis. Classes of phospholipids typically found on the inner face of biomembranes include phosphatidylinositol (PI). The generation of phosphoinositides derived from PI via phosphorylation events will generate classes of important lipids enrolled in cell signaling and phagocytosis as example of phosphatidylinositol (4)-phosphate (PI(4)P=PIP), PI(5)P, PI(4,5)P2 (PIP2), PI (3,4)P2, and PI(3,4,5)P3 (PIP3). As mentioned previously in this chapter, these phosphoinositides, especially PIP2 and PIP3, are capable of binding and increasing the activity of proteins that modify membrane chemistry and the actin cytoskeleton. As an example, PIP2 increases the activity of WASP, a protein that stimulates actin polymerization via Arp2/3.
This class of PIs in addition to their relevance in particle internalization is important during the phase of phagosome maturation into a degradative compartment, the phagolysosome. In phagosomal membranes PIP2 activates the actin nucleators of the Ezrin, Moesin, and Radixin family inducing polymerization of F-actin and therefore phagosome maturation . This will be addressed later in this chapter in the context of the manipulation of the actin cytoskeleton by pathogens in order to establish an intracellular niche.
2.2. Induced phagocytosis by invasive pathogens
Classically, the manipulation of the actin cytoskeleton by invasive pathogens was classified into two general mechanisms according to the type of morphological changes that occur in the host cell—the zipper and trigger phagocytosis . Entry of uropathogenic
Adherence to nonprofessional phagocytic cells, epithelium by a pathogen is necessary to avoid mechanical clearance and is the first step of colonization by for example enteropathogens. Thus bacterial pathogens exhibit a large variety of cell surface adhesins, including fimbriae (pili) and afimbrial adhesins some of which participate in the internalization step. Likewise, in this type of entry, a bacterial adhesin binds to a host cell surface receptor involved in cell-to-cell adhesion and/or activates regulatory proteins that modulate cytoskeleton dynamics. Moreover, adherence and internalization into epithelial cells looks to be a strategy used by pathogens to escape destruction by immune cells as described below.
Most type I pili expressed by pathogenic
Curiously the vaccinal strain for tuberculosis
In endothelial cells, the T4SS-pilus-mediated adhesion of
2.3. Macropinocytosis, induced macropinocytosis, and coiling phagocytosis
Unique molecular properties associated with the process of macropinocytosis are beginning to be elucidated. Because of their size and the fact that they may be formed without activation by ligands, the large vacuoles (macropinosomes) formed during this pinocytosis event can contain extracellular fluid and pathogens. At the mechanistic level, phagocytosis and macropinocytosis present many similarities including the involvement of phosphoinositol phosphate signaling and actin cytoskeleton reorganization. During macropinocytosis it is not observed a direct connection between bacteria/cargo and multiple receptors but it was demonstrated the relevance of tyrosine kinase receptors involved in responses to growth factors as the epidermal growth factor and platelet-derived growth factor. The consequence of intensive actin remodeling results in ruffling protrusions at the cell surface, or in unilateral large pseudopodia formation leading to the formation of large macropinosomes. Activated receptor tyrosine kinases, as well as the Src family kinases, are clearly observed on newly formed macropinosomes. Therefore in concert with the morphological definition provided by Lewis in 1931 based on ruffling formation, and elevation in response to growth factor stimulation can be used to define macropinocytosis .
Macropinocytosis has been observed in professional phagocytes as well in epithelial cells. Immature dendritic cells and activated macrophages display high levels of constitutive macropinocytosis . The consequent internalization of large volumes of extracellular solute that accompanies macropinocytosis facilitates their capacity to continuously survey the extracellular space for foreign material. In fact, this increased levels of macropinocytosis upon encounter with the antigen/pathogen enhances both antigen capture and antigen presentation by dendritic cells as well as the complete clearance of pathogens after macrophage activation by inflammatory stimulus .
In epithelial cells, an induced form of macropinocytosis was observed after infection with pathogens such as
Invasive enteropathogens, such as
In comparison, the effectors IpaC, IpgB1, and VirA of
Recently, the mechanism of
Surface-adherent pathogens, such as enteropathogenic or enterohaemorrhagic
Patients with inflammatory bowel disease exhibited an increased number of mucosae-associated
Alveolar macrophages constitute the main defense against
Coiling phagocytosis is an actin dependent endocytic event, morphologically accompanied by a typical pseudopodia that looks like whorls or wrapps around the bacteria in several turns (Figure 1). A definition of the phenomena is complex as it presents similarities to macropinocytosis and conventional phagocytosis: for the first due to the large pseudopodia; for the second due to cargo specific entrapment. In coiling phagocytosis, the single pseudopodia do not trap fluid droplets but enclose microbes; however, the multiple pseudopod whorls have largely self-apposed surfaces instead of those that are microbe-apposed surfaces.
In summary, deciphering the players that induce or prevent phagocytosis in one infection context may be used as strategies to clear pathogens in other context. It is an interesting observation that preinfection of cultured gastric cells with yersinia expressing Yop virulence factors that interfere with the same signaling events, impaired phagocytosis of
Define what receptors stimulate to induce a more bactericidal response of infected cells, how to control bacterial load that is internalized to induce apoptosis, as is the case of microRNAs that control WASP in tuberculosis context; how to neutralize factors that prevent Rho family of GTPases to modify actin in order to induce phagocytosis of extracellular pathogens, these are a few targets to explore deeply. Other relevant area to act is how to neutralize bacterial adhesins, secretion systems or their access to surface receptors as integrins to prevent epithelia invasion. It is imperative to decipher what are the virulence factors that mimics or induce growth factors that leads to induced macropinocytosis. In addition, it is important to find how to neutralize secretion systems that reorganize the actin cytoskeleton for macropinosome formation and therefore for pathogen invasion of epithelial and endothelial cells, important reservoirs of latent infections.
3. Acting on actin for the establishment of an intracellular niche
In addition to particle binding and internalization, phagocytosis includes the process of phagosome maturation leading to pathogen destruction in the acidic hydrolytic environment of the phagolysosome. These events are important innate immune mechanisms. Indeed a consequence of phagosome maturation is the activation of the antigen presentation machinery. Macropinocytosis culminates in the appearance of a large vacuole that, indeed follows the fate of the phagosome. Some pathogens have evolved to establish sustained infection in professional phagocytes preventing phagosome maturation as is the case of
The material in endosomes or phagosomes that is destined for lysosome degradation by endocytosis or phagocytosis reaches this compartment by fusing with the organelle. Critical for this is the membrane composition of the correct repertoire of lipids, membrane-bound proteins, and also proteins that shuttle on and off membranes. The manipulation of the phagosomal membrane by pathogens may block the ability of fusion with lysosomes leading to a vacuole that may be trafficked apart from the endocytic route. In alternative, the vacuole may be arrested from maturation along the endocytic pathway by pathogen membrane manipulation leading to continuous transient fusion events with upper compartments.
Phagosome maturation is known to be influenced by the lipid species present on the outer and most likely inner membrane, and published studies have focused mostly on kinases that generates PIP, and PIP2, which binds actin nucleation proteins . Additionally, the ability to nucleate actin leading to F-actin polymerization from phagosomal membranes was associated to the formation and availability of actin tracks for organelles to move towards the actin-nucleating source, increasing vesicle trafficking, fusion events, and phagolysosome biogenesis (Figure 1) . Identifying key roles for PIP and PIP2 opened the door for the analysis of several other lipids that interconnected with these phosphoinositides in the actin assembly process, as well as sphingolipids and fatty acids favouring phagosome maturation [11, 51]. Examples of F-Actin stimulatory factors includes the eicosanoide omega 6 arachadonic acid, ceramide and sphingosine-1-phosphate.
Several groups have explored the role of actin cytoskeleton during
Another pathogen that blocks phagosome maturation is
4. Acting on actin for pathogen dissemination: actin-based motility of pathogens and innate immunity
Early after host invasion some pathogens escape lysosomal destruction and antigen presentation by escaping into the cytosol. Thereafter, actin polymerization is manipulated by several cytosolic pathogens such as
When intracellular moving bacteria reaches the plasma membrane, they push out long protrusions that are taken up by neighboring cells, facilitating the infection to spread from epithelial cell to cell in the absence of immune surveillance. At the cell-to-cell cytoplasmic membranes sites, the cytosolic actin-based moving pathogens induce the formation of surface protrusions that force the internalization from the infected cell into noninfected neighbor cells. The process of engulfment is called paracytophagy and involves internalization of a double membrane containing pathogen: the inner from the donor cell and the outer from the recipient cell (Figure 1) [54, 55]. At this point the pathogen may escape again to cytosol to start a new infection process.
In the case of enterophatogenic
The actin-based motility of
The predominance of a membrane surrounding vacuole during the infection of most intracellular pathogens looks to be related to immune protection from the defensive mechanisms that exist in the cytosol. The arrival of a pathogen or their PAMPs to the cytosol could “wake up” several patrol mechanisms that include cytosolic PRRs. The sensing by cytosolic innate receptors leads to an inflammatory response by secretion of proinflammatory cytokines and chemokines or a interferon type I response that overall leads to antimicrobial response; the stress in the cytosol induce inflammasome assembly .
Therefore, the arrival of the pathogens in the cytosol establishes a bridge to the innate immune response by contact of the pathogen-associated molecular patterns (PAMPS) with PRRs, such as NLRPs (Nod like, similar to Toll like receptors- TLRs on cell membranes). Additionally, and by causing cytosol stress, PAMPS will activate (via PRRs) the inflammasome, a complex structure of proteins similar to the apoptosome . Inflammasome assembly will lead to pro-Interleukin1β (pro-IL-1β) and pro-IL-18 inflammatory cytokine activation via caspase 1 and to the programmed cell death dependent on caspase 1, as it is pyroptosis and pyronecrosis . This is a natural immune response in gut and respiratory epithelial cells but not in endothelial vascular and lymphatic cells that lakes these cytosolic receptors and constitutes important host niches for intracellular pathogen survival [33, 47].
Rickettsiae possess a tropism to endothelial cells, a tissue that usually serves as barrier to intravascuolar blood from surrounding tissues. This tropism leads to the endothelial cell injury associated with complications of the disease. RickA (mentioned previously in this chapter) is a protein present in the pathogenic species
Macrophages, in contrast to endothelial cells, possess NLRs and other PRRs families. During
The detection of cytosolic LPS, as a consequence of disruption of replication vacuoles harboring Gram-negative bacteria was shown to trigger the activation of murine caspase-11 that leads to the assembly of a noncanonical inflammasome . Caspase-11 (Casp-4 in humans) is also crucial for clearance of bacteria that escape the vacuole, such as
Another potent host defense mechanism that restricts intracellular pathogens is autophagy. Some intracellular bacteria cause the formation of ubiquitinated aggregates around either bacterial structures or replication vacuoles, and the autophagic machinery can recognize these. The process of bacterial clearance by selective autophagy is called xenophagy.
All together these findings let us to postulate that important strategies to fight pathogens will pass by control their life cycle in the cytosol. Either addressing the linkage of actin tails to Arp2/3 or WASP proteins or neutralizing the bacteria actin nucleators to prevent motility and spread to neighbor cells; either to induce death of the infected cell by apoptosis, pyroptosis, or necrotic lysis; either by exposition of pathogen signatures that leads to xenophagy; altogether these are a few potential strategies to address in the future.
5. Concluding remarks
During evolution, higher eukaryotic organisms have developed epithelial barriers and phagocytic immune cells to resist and fight infections. The discovery of antibiotics in the early part of the last century led to predictions that bacterial infections would be kept under tight control via natural systems and treatment with drugs. But the capacity of bacteria to evade natural protective systems and rapidly develop resistance to antibiotics had led to the current situation of bacteria posing major health problems in both the developed and underdeveloped world. There is now a major requirement to find alternative treatments to fight bacterial pathogens. Over the years, various studies have elucidated the mechanisms by which bacterial PAMPs, adhesins, and secretion systems together with their translocated effectors target and alter the host actin dynamics. Targeting the host actin machinery is important for the survival and pathogenesis of several extracellular, vacuolar, and cytosolic bacteria. Studying the manipulation of host actin by pathogens has vastly improved our understanding of various basic cell biological processes in host cells while giving key insights into both bacterial pathogenesis and host innate immunity. Together this opens a new and exciting field of research with the objective of discovering new classes of antibiotics that directly or indirectly interfere with this actin-modulating mechanism.
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